Genome-based surveillance reveals cross-transmission of MRSA ST59 between humans and retail livestock products in Hanzhong, China

Methicillin-resistant Staphylococcus aureus (MRSA) has been recognized in hospitals, community and livestock animals and the epidemiology of MRSA is undergoing a major evolution among humans and animals in the last decade. This study investigated the prevalence of MRSA isolates from ground pork, retail whole chicken, and patient samples in Hanzhong, China. The further characterization was performed by antimicrobial susceptibility testing and in-depth genome-based analysis to identify the resistant determinants and their phylogenetic relationship. A total of 93 MRSA isolates were recovered from patients (n = 67) and retail livestock products (n = 26) in Hanzhong, China. 83.9% (78/93) MRSA isolates showed multiple drug resistant phenotype. Three dominant livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) sequence types were identified: ST59-t437 (n = 47), ST9-t899 (n = 10) and ST398 (n = 7). There was a wide variation among sequence types in the distribution of tetracycline-resistance, scn-negative livestock markers and virulence genes. A previous major human MRSA ST59 became the predominant interspecies MRSA sequence type among humans and retail livestock products. A few LA-MRSA isolates from patients and livestock products showed close genetic similarity. The spreading of MRSA ST59 among livestock products deserving special attention and active surveillance should be enacted for the further epidemic spread of MRSA ST59 in China. Data generated from this study will contribute to formulation of new strategies for combating spread of MRSA.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is one bacteria type that may cause numerous clinical manifestations ranging from mild skin and soft tissue infections to lifethreatening fulminant invasive diseases (Turner et al., 2019;Tuffs et al., 2022).MRSA has been recognized in hospitals, community and livestock animals and the epidemiology of 10.3389/fmicb.2024.1392134MRSA is undergoing a major evolution among humans and animals in the last decade (Chen et al., 2021;Yu et al., 2021).Since the livestock-associated MRSA (LA-MRSA) was first recognized in Europe in 2003, LA-MRSA has been identified in numerous countries around the world, including China (Cui et al., 2009;Silva et al., 2023).Multilocus sequence typing (MLST) analyzes seven constitutively expressed (housekeeping) genes that are essential to cellular functioning of organisms.LA-MRSA of sequence type (ST) 398 dominates in Europe, Australia and the United States, while LA-MRSA-ST9 is the main epidemic lineage in Asia (Yu et al., 2021;Silva et al., 2023).Several studies have identified specific LA-MRSA within the community acquired-MRSA category (Bisdorff et al., 2012;Mascaro et al., 2018;Sun et al., 2019).Recently, LA-MRSA-ST398 and other sequence types previously widely disseminated among human beings (such as ST59) were also identified in livestock animals and meat samples from China (Wang et al., 2014;Li et al., 2021;Zhang et al., 2021).
Because of the huge volume of livestock animals and the consumption of livestock products in the community, there is a concern that MRSA may be spreading and concentrating in livestock animals with subsequent dissemination into the community population through contact with livestock, farm environment or retail livestock products (Yu et al., 2021;Li et al., 2022).Especially retail livestock products, such as pork and poultry, are susceptible to MRSA contamination during slaughtering process and can become an ideal media of MRSA transmission to the kitchens (Hennekinne et al., 2012;Li et al., 2021).
In 2009, LA-MRSA-ST9 was firstly recognized from the swine and farm worker samples in Hanzhong city, Shaanxi province and all MRSA isolates in the previous study were grouped into ST9-spa899 (Cui et al., 2009).Hanzhong city lies in the center of the Hanzhong Basin.The south of the city is the Daba mountains and the north of the city is the Qinling mountains which limits the human migration and economic exchange with other areas.The location factor makes this city an ideal place for LA-MRSA transmission study.Since the pilot study in 2009(Cui et al., 2009)), no follow-up study was conducted on the transmission of LA-MRSA in this area.
The objective of this study was to determine the prevalence of MRSA isolates in ground pork, retail whole chicken and patient samples in Hanzhong city.The isolates were further characterized by in-depth genome-based analysis to identify the resistant determinants and their phylogenetic relationship.

Sample collection and MRSA isolation
From July 2019 to May 2020, retail ground pork (n = 88) and retail whole chicken carcasses (n = 87) were collected from seven supermarkets in Hanzhong, China.Each supermarket was visited once a month.On each sampling day, no more than two whole chicken carcasses or two ground pork samples were randomly selected from each sampling site.All samples were transported to the laboratory and processed within 4 h.Each whole chicken carcass was immediately aseptically removed from the package and placed in a 3500 stomach bag (Seward, UK) followed by the addition of 500-mL buffered peptone water (BPW; Becton-Dickinson, Beijing, China).The bag was manually massaged for 3-5 min and the rinse were used for MRSA isolation.25 ml of the rinse or 25 g of ground pork samples were added into 225 ml enrichment broth containing 1% tryptone, 7.5% sodium chloride, 1% mannitol and 0.25% yeast extract and incubated at 35 ± 1 • C.After 22-24 h incubation, a loopful of the culture was inoculated onto selective MRSA agar plates (BBL CHROMagar MRSA) and incubated at 35 ± 1 • C for 24-48 h.Purple colonies on the selective plates were screened for coagulase activity.All MRSA isolates were confirmed by the API Staph ID test (BioMe rieux, Beijing, China) and PCR screening for the carriage of nuc and mecA (Merlino et al., 2002).One confirmed MRSA isolate from each sample was selected for further study.All MRSA isolates were kept in brain heart infusion broth (BD, China) with 50% glycerol at −80 • C freezer for further analysis.

Collection of MRSA isolates from patients
MRSA isolates from patients were collected from the 3201 hospital which is the largest hospital in Hanzhong, Shaanxi, China.During the food sample collection, this hospital tested 7033 independent blood samples, 3517 cerebrospinal fluid samples, 128 wound secretion samples from inpatients for bacteria infection and 406 fecal samples from outpatients for S. aureus.The MRSA isolates from outpatient and inpatient samples were obtained and included in this study.

Genome sequencing and assembly
Bacterial genomic DNA of each isolate was extracted from 2 mL fresh culture using QIAamp DNA Mini Kit (Qiagen, Germany) following manufacturer's instructions.DNA was subjected to quality control by visualizing electrophoresis products on a 1% agarose gel and quantifying them using a Qubit fluorometer (Invitrogen, Shanghai, China).WGS was performed with massively parallel sequencing (MPS) Illumina technology at Beijing Novogene Bioinformatics Technology Co., Ltd.A paired-end library with a 350 bp insert size was constructed and sequenced by Illumina NovaSeq using PE150 strategy.Illumina PCR adapter reads and low-quality reads were filtered by Readfq (version:10) and the filtered reads were assembled using SOAP denovo (version 2.04), SPAdes (version 3.10.0)and Abyss (version 1.3.7) to generate scaffolds (Li et al., 2008;Simpson et al., 2009;Bankevich et al., 2012) which were integrated by CISA software (Lin and Liao, 2013).The initial assembly results were optimized and matched using Gapclose (version 1.12) software to obtain the final assembly results (Luo et al., 2012).WGS data of 93 MRSA isolates were deposited into GenBank under BioProject accession number PRJNA966921 (Supplementary Table 3).

Statistical analysis
The chi-square test was used to determine differences in the resistance rate of S. aureus.All statistical analyses were performed using the SPSS 18.0 software package.
After SNP analysis, eight isolates harbored lukF/S-PV were grouped into one cluster, comprising isolates 5-23, 5-19, 7-20, Y3, 5-31, Y13, 7-4 and 7-32 (Figure 1).Human isolates from blood, fecal and wound secretion samples were crossly distributed.A cluster of five ground pork isolates (isolates 2884, 3115, 3116, 3117 and 3118) of CLI-ERY-OXA-TET multidrug resistant phenotype was identified.Two ground pork isolates were scattered among patient isolates with isolate 1-22 from ground pork showing a close relationship with isolate 7-5 from patient blood sample, while another ground pork isolate 2885 showed a close relationship with pork isolate 3025 of online reference.Six isolates from whole chicken carcasses were scattered among human isolates.Genetic similarities were found among five pairs of chicken and human isolates (Figure 1).

Virulence genes among ST59, ST9 and ST398 isolates
All ST59, ST9, and ST398 isolates were found to contain the following virulence factors, including the metalloprotease (aur) and the hemolysins hlgA, hlgB, and hlgC.PVL was present in eight ST59-spa t437 human isolates (8/44, 18.2%) and six isolates were staphylokinase (sak) negative, but all the PVL negative ST59 MRSA isolates were sak positive.The distribution of scn, sak and enterotoxin encoding genes showed different patterns across different STs.All ST59 (n = 57) and patient ST398 (n = 4) isolates were scn positive, all ST9 (n = 10) and the food ST398 (n = 3) isolates were scn and sak negative.
Among ST59 isolates, sak was identified among 51 isolates and enterotoxins seb, sek and seq were identified among 50 isolates.Both sak and enterotoxins seb, sek and seq were identified among 45 isolates from ground pork (n = 7/7), whole chicken (n = 5/6), fecal swabs (n = 13/15), sterile sites (n = 10/15) and wound secretion (n = 10/14) samples.All ST9 isolates harbored enterotoxin gene seo, and eight ST9 food isolates also harbored more enterotoxin gene (seg, sei, sem, sen and seu), but these genes were absent in ST398 isolates (Supplementary Table 1).Phylogenetic relationship of ST59 isolates from retail livestock products and patients, Hanzhong, China.The genome sequences of 65 ST59 isolates were aligned through SNP analysis, including 8 online isolates.The information of online isolates was provided in Supplementary Table 4.

Characterization of the remaining MRSA isolates
Except one isolate recovered from the ground pork sample, the other leftover MRSA isolates (n = 18) were recovered from patient samples.These 19 isolates were grouped into eight STs.All ST239 (n = 9) isolates showed multiple resistant phenotypes and carried SCCmec_type_III(3A) and were grouped into spa t030 (n = 5), spa t459 (n = 4) types.SCCmec_type_IV (n = 8) and SCCmec_type_V (n = 3) were identified among the other isolates.Except one ST45 isolate from the fecal sample, the other MRSA isolates (n = 18) were all scn and sak positive.lukE/D was present in 15 isolates.

Discussion
From patients and retail livestock products in Hanzhong, China, three dominant LA-MRSA sequence types were identified: ST59-t437 (n = 47), ST9-t899 (n = 10), and ST398 (n = 7).There was a wide variation among sequence types in the distribution of tetracycline-resistance, scn-negative livestock markers and virulence genes, indicating different origins and evolutionary processes.We found that in Hanzhong, China, a major human MRSA ST59, not LA-MRSA ST9 or ST398, became the predominant interspecies MRSA sequence type among humans and retail livestock products.Some MRSA-ST59 isolates from patients and livestock products showed close genetic similarity.Our study further indicated that certain human MRSA may become LA-MRSA and transfer from humans to livestock animals or vice versa, which demonstrated the importance of continuous MRSA surveillance among humans and livestock animals.
Our data showed ST59 MRSA was dominant not only among patients in Hanzhong (44/67), but also among livestock product isolates (13/26).Initially being reported in North America in the early 2000s (Enright et al., 2002), ST59 MRSA has gradually been replacing ST239 and ST5 human clones and become the predominant sequence type in most hospitals of China since 2010 (Li et al., 2018;Jin et al., 2021;Wang et al., 2022).However, ST59 MRSA remains geographically confined and is low prevalence in Europe and North America (Pimentel de Araujo et al., 2021;Di Gregorio et al., 2023).Previous studies have shown ST9 is the dominant LA-MRSA in China and other Asian countries as opposed to ST398 in Europe and North America (Silva et al., 2023).Recent studies have also reported ST59 MRSA isolates Phylogenetic relationship of ST9 isolates from retail livestock products and patients, Hanzhong, China.The genome sequences of 18 ST9 isolates were aligned through SNP analysis, including 8 online isolates.The information of online isolates was provided in Supplementary Table 4.
were scattered among livestock animals in China (Wang et al., 2021;Li et al., 2022), our data indicated that the detection rate of ST59 in livestock products in Hanzhong was higher than ST9 and 398 isolates.ST59 MRSA isolates displayed a greater overall phylogenetic diversity than LA-MRSA ST9 isolates did which indicated the multiple origins of ST59 isolates or higher adaptability within different hosts, suggesting that the ST59 isolates might undergo multiple and continuous evolutionary events.Usually, ST59 isolates carry SCCmec_type_IV (55/57) or SCCmec_type_V, both are smaller SCCmec cassettes that may reduce the host fitness burden.Coculture experiment in a previous study showed that ST59 isolates displayed higher growth rates and competitive capacity than MRSA ST239 in vitro which provided further evidence that ST59 clones may have a better capacity for surviving outside the host and promote ST59 transmission among livestock animals and human beings (Li et al., 2018).In this study, some ST59 isolates from livestock products were scattered among patient isolates in phylogenetic tree suggesting that frequent exchanges might occur between livestock animals and patients.Because of the higher adaptability and competitive power of ST59 isolates, a detailed study of the genetic basis for the successful dissemination of ST59 among both humans and livestock animals in China should be conducted.
Multiple livestock-association markers, such as scn-negative, tetracycline-resistance, CC9, CC398 have been reported in different studies (Verkaik et al., 2011;Cuny et al., 2015).There is growing evidence that accessory genes carried by prophages of S. aureus significantly modulate bacterial fitness as they carry multiple virulence factors (VFs).These VFs include human immune evasion cluster (IEC) comprising the genes sak, chp, scn and sea/sep (Nepal et al., 2021).As a marker of IEC, scn has been recognized as the indicator of human S. aureus isolates and might be useful for differentiating livestock isolates from human isolates (Rinsky et al., 2013).In this study, all human ST398 (n = 4) isolates were scn positive, but all ST9 (n = 10) and food ST398 (n = 3) isolates were scn negative.This was consistent with a recent study that the loss of IEC might happen after their shift from human to animals because antimicrobials in feed can induce scn prophage loss (Allen et al., 2011;Price et al., 2012;Yu et al., 2021).All food (n = 13) ST59 isolates in this study were scn-positive which indicated these isolates might jump from human into the livestock animals recently.A recent study also found scn-positive ST59 was Phylogenetic relationship of ST398 isolates from retail livestock products and patients, Hanzhong, China.The genome sequences of 15 ST398 isolates were aligned through SNP analysis, including 8 online isolates.The information of online isolates was provided in Supplementary Table 4.
the major MRSA among Yak (Bos grunniens) herds in Tibetan, China (Zou et al., 2022).A continuous surveillance study should be carried out to find out how long ST59 isolates would keep scn after it was transmitted to livestock hosts and the factors that might influence the speed of scn loss.
Tetracycline-resistance is another livestock-association marker different from human isolates (Rinsky et al., 2013).Tetracyclines are a class of broad-spectrum antibiotics, including naturally occurring and semi-synthetic tetracyclines (Roberts, 2003).Due to their side-effects, the use of tetracyclines is limited in human clinics, but they are still been used globally as important infection treatment measures and growth promoters among livestock animals which promoted the tetracycline resistant determinants transmission in livestock products (Inglis et al., 2019;Chen et al., 2023).Since animals cannot fully absorb or metabolize tetracyclines, they excrete a significant fraction of such drugs or of their breakdown products into the environment via feces or urine which may also promote the transmission of resistant determinants (Mackie et al., 2006).More than 30 tetracycline specific resistant determinants have been recognized and some of them can be transferred horizontally among different bacteria through mobile genetic elements, such as plasmids, phages or integrons (Roberts, 2003).In this study, the food isolates (20/26) showed significantly higher frequency of tetracycline resistance than isolates from human beings (27/67) which indicated the food isolates circulating among livestock animals for considerable time.The genotypes of tetracycline resistant determinants were corresponding to the sequence types which further indicated different evolution processes of these isolates.
Genetic advantages might contribute to the replacement of LA-MRSA from ST9 to ST59 locally.Diversified genetic context was found among 57 ST59 MRSA isolates which were group into six spa types with t437 (n = 47) as the local dominant LA-MRSA type which was more diversified than ST9 (n = 10) isolates that were all belonged to spa t899 (Cui et al., 2009;Wang et al., 2017).Other studies also reported much more diverse genetic context of ST59 isolates that probably reflected active and extensive genetic recombination of this clone (Jin et al., 2021;Wang et al., 2021).
The diversified genetic context might have more exchange and adaption opportunities and speed up the transmission of this clone.More virulence factors might be another advantage that could contribute to ST59 higher transmission compacity than ST9 isolates (Li et al., 2016).ST9 was originated from human-adapted strains which had lost genes related to the evasion of the immune system (Yu et al., 2021).In this study, all ST9 (n = 10) isolates were scn and sak negative which further confirmed this theory.
There are several shortages of this study.All MRSA isolates were from patients and retail livestock product samples in Hanzhong city, China and no isolates recovered from livestock animals were included.Limited isolates of ST9 and ST398 were analyzed from human and retail livestock product samples in this study.More isolates from other areas should be analyzed to confirm the distribution patterns found in this study.

Conclusion
Because of the high adaption and transmission capacity, MRSA ST59 may be widespread and become the dominant clones among livestock animals in the future.Therefore, active surveillance should be enacted for the further epidemic spread of MRSA ST59 in China.